Analytes also include, among other things, low-molecular-weight pollutants, e.g., pesticides and their decomposition products (metabolites). These pollutants may also be contained in aerosols.
In recent years, public discussion has increasingly frequently focussed on the use of synthetic pesticides for the purpose of plant protection and the environmental pollution associated with it. The European Drinking Water Ordinance, the "EC Guidelines for the Quality of Water for Human Consumption," as well as the MAC Values List (the MAC value is the maximum allowable concentration of a working substance in the form of a gas, vapor or suspended matter in air at the workplace) specify limit values for these substances. The limit value is, e.g., 0.1 .mu.g per L of water for certain substances. The detection limit should be below this value by one to two orders of magnitude for accurate analysis. The large number of substances in question and the low limit values represent a big problem for physical and chemical analysis.
Physical and chemical analytical methods require complicated enrichment processes, are highly expensive, time-consuming, and can be carried out only by specialized skilled technicians in specially equipped laboratories. To avoid the above-mentioned disadvantages, immunological processes--which have already been used routinely in clinical diagnostics for the detection of a great variety of substances--have recently been developed, especially for the quantitative and qualitative determination of pollutants in soil, water, or air samples (e.g., West German Offenlegungsschrift No. DE-OS 40 13 004). DE-OS 40 13 004 describes a classical immunochemical method, used for low molecular weight organic substances, e.g. haptens, encountered in the field of analytical methods for pollution control. To this effect, DE-OS 40 13 004 proposes to use an antibody-enzyme-conjugate in conjunction with a competitive immunoassay.
Immunoassays are highly sensitive test systems for the qualitative and/or quantitative determination of substances on the basis of the antibody-antigen reaction, wherein the substance to be detected (analyte) acts as an antigen. The antibodies are first induced by immunizing laboratory animals, and are subsequently isolated according to conventional methods. These so-called "polyclonal" antibodies are highly heterogeneous in terms of both their specificity and affinity with respect to the analyte and their association with the individual classes of immunoglobulins. Instead of individual immunized animals, it is also possible to use hybrid somatic cell lines (hybridomas) as the source for so-called "monoclonal" antibodies (mAbs) against very specific antigens (=analytes).
The advantages of monoclonal antibodies over polyclonal antibodies are numerous, e.g.,
a) monoclonal antibodies (mAbs) can be obtained in large amounts and at a high degree of purity; PA0 b) the mAbs are homogeneous in terms of the antigen reactivity, and their properties are the same in each batch prepared; PA0 c) hybridoma cell lines which produce mAbs can be stored for several years, without losing their specific properties. PA0 a) A microtiter plate normally consists of 96 depressions, which is meaningful for a larger serial investigation, but it represents a waste of material in the case of individual samples; PA0 b) due to the fact that the depressions located adjacent to each other in the microtiter plate are normally coated with only one kind of antigen or antibody, rapid differential diagnosis of pollutants is possible only with difficulty; PA0 c) the binding of the antigens and antibodies is again partially broken during the individual reaction steps, especially due to the intermediate rinsings, so that up to 70% of the original antigen-antibody complex may be lost. As a result, there is only a limited possibility of obtaining standardized and reproduced results; PA0 d) the limited adsorption capacity of these plastic surfaces represents another disadvantage during the antigen or antibody loading of the microtiter plate.
Monoclonal antibodies are therefore preferably, but not exclusively, used for the present invention.
The antigen-antibody binding is used for detecting a pollutant in immunoassays. The same intermolecular forces are responsible for the formation of the antigen-antibody complexes both in vivo and in vitro. They are based on the attraction of groups carrying opposite charges, on the interaction between hydrophilic and hydrophobic groups, as well as on the van der Waals forces, which originate from the interactions between electron clouds. All these interactions or bindings are, in principle, reversible and non-covalent, and the forces exert their effect only when the antibody and antigen come very close to each other.
Homogeneous and heterogeneous systems are distinguished in the immunoassays. In the homogeneous system, the enzyme activity, e.g., that of a hapten-enzyme conjugate, is influenced by the antigen-antibody reaction and is measurable. Washing and separation steps are not necessary. Systems in which the enzymatic activity of the conjugate is not influenced require one or more separation or washing steps, and are therefore called heterogeneous tests. The heterogeneous immunoassays include, e.g., competitive assays. Such test methods involve, in general, the competition between a known amount of labeled analyte and an unknown amount of unlabeled analyte to be determined for a limited amount of an analyte-specific, solid phase-bound partner of an immunological reaction. The solid phase-bound, labeled analyte can be separated from the free, labeled analyte after the reaction, and its amount can be determined chemically or physically on the basis of the labeling. The measured value is inversely proportional to the amount of the unlabeled analyte to be determined. As an alternative, it is also possible to determine the amount of the labeled analyte that has remained non-bound. The measured value is directly proportional in this case to the amount of the analyte to be determined. Examples of such competitive, heterogeneous immunoassay systems are radioimmunoassays (RIAs), fluorescence immunoassays (FIAs) or enzyme immunoassays (EIAs), as well as enzyme-linked immunosorbent assays (ELISAs). The basic principle is the same in all these methods of detection. The most important difference is in the use of different labels. Heterogeneous immunoassays are very often performed by bringing together the individual reaction partners and reagents manually, separating the solid phase from the liquid one manually, and determining the amount of label, if necessary, after further washing and incubation steps.
The principle of an immunoassay, which is based on the displacement of one of the reaction partners of an immunological reaction by another, has been known from the technical literature (e.g., WO 91/13354). Mixing of the sample with the tracer (it is defined as an analyte or analyte derivative, to which one or more labeling elements are bound), which is necessary in competitive assays to start the competition reaction for the limited antigenic binding sites on the carrier, is eliminated in this displacement immunoassay. Separate reagent vessels or reagent layers along with the corresponding reagents are also eliminated in this displacement assay. The displacement takes place in a reaction column of a flow injection device. The analyte displaces during the reaction the tracer, which is labeled with, e.g., fluorophores, from its binding with the specific antibodies, which are bound to a matrix of the reaction column (i.e., the solid phase). Since no reagents are used in this embodiment, a fluorescence-measuring instrument must be used to quantitatively determine the amount of tracer molecules displaced. Since exactly one tracer molecule is displaced per molecule of analyte, there is a directly proportional relationship between the analyte concentration and the measured signal in displacement immunoassay. Even though an immunoassay using fluorophores as the labeling element makes it possible to obtain quantitative data on the reaction, it does require complicated and expensive measuring means, and is therefore unsuitable for performing a rapid screening on the spot for the presence of pollutants and for identifying their nature and amount.
Vessels have been known for performing the immunoassays, which are called microtiter plates and consist of a plurality of depressions arranged in a plastic card (e.g., DE 91 00 320 U1). These microtiter plates are coated with the antibodies or antigens prepared according to a complicated process. All further incubation and washing steps will subsequently take place in the above-mentioned depressions. However, this process, in which the reaction vessel also constitutes the carrier coated with the antibodies or antigens, has several disadvantages:
Immunological test kits, e.g., pregnancy tests, in which small, dyed plastic particles ("latex beads") are used as labels on antibodies, have been known as well (e.g., DE-OS 40 37 736 or DE-OS 40 37 724). DE-OS 40 37 736 and DE-OS 40 37 724 each shown an example of a so-called sandwich-assay with a special aspect to dyed particles-sandwich-assays or bead-migration-assays or chronomatographic-strip-assays which are commonly used for pregnancy test procedures. DE'736 especially relates to a dip stick assay, whereas DE'724 concerns a migration-type test as disclosed on page 8, line 5 to page 9, line 4 of our description. The test carriers are special thin layers or membranes, which permit the "migration" of the antibodies thus labeled on the carrier layer toward another immunological binding partner. The driving forces of this migration include the capillary suction forces of the carrier material. The immunoassay principle of the test is the "non-competitive" assay. The antibody immobilized adsorptively or covalently on the solid phase can be reacted with a sample of unknown analyte concentration according to this process, which is generally also called "sandwich method," here specifically "dyed particles sandwich" assay, "bead migration" assay, or "chromatographic strip" assay. The two reaction partners are contacted via a migration process of the liquid phase with the dissolved analyte, or via the carrier. A second antibody (antibody conjugate), which is specific of the analyte and is also bound to a label, is added, and this second antibody will be bound to the analyte already fixed on the carrier. The intensity of the signal, which can now be evaluated optically, is an indicator of the amount of analyte in the sample. This antibody conjugate is preferably placed on the carrier, still in front of the zone with the covalently bound, unlabeled antibody. This antibody conjugate can thus be bound by the immobilized, unlabeled antibody after migration of the analyte together with it as antibody conjugate-antigen complexes. If no analyte is present in the sample, no visible complexes will form from antibody conjugate+immobilized antibody.
Such test methods have been established for larger multivalent or polyvalent analytes. However, these tests provide only qualitative measured results, which is sufficient and meaningful in the case of use as a pregnancy test or for determining infections in medical diagnostics. Qualitative measurement of smaller, environmentally relevant pollutant molecules and their quantitative determination are not possible in this manner.
WO 91/12528 discloses an immunoassay in the form of a test strip provided with a plurality of zones. To detect an analyte occurring in an aqueous solution, the test strip is dipped into this solution. The solution will then migrate through the strip due to capillary force, and reach first a first zone, which contains a first, labeled antibody that is specific of a first binding site of the analyte. The solution continues to migrate to a second zone, which contains a second antibody that is specific of a second binding site of the analyte. The sandwich consisting of the first antibody, the analyte, and the second antibody continues to migrate with the solution to a third zone, in which the sandwich is trapped, and the labeling of the first antibody is thus concentrated and thus made visible.
The disadvantage of this immunoassay is that two antibodies are needed for two different binding sites of the analyte. This is often impossible in the case of small analyte molecules.
FIG. 13 of the drawings shows schematically a process described in EP-PS 191 640, which operates with a test strip 100 consisting of absorbent material, on which a first antibody 102 is immobilized in a zone 101. One end of the test strip 100 is dipped into a test solution 103, which contains the analyte 104 to be determined and a second antibody 105, which is specific of the analyte 104. The first antibody 102 binds the complex consisting of the analyte 104 and the second antibody 105 during the capillary migration of the test solution 103 through the test strip 100. The complex bound to the first antibody 102 is dyed and detected as a result (not shown in FIG. 13) by means of a developing solution.
The disadvantage of this process is that two specific antibodies 102, 105 are needed.
The first antibody 102 must be able to distinguish between a second antibody 105 with and without bound analyte 104. This is hardly possible in the case of small analyte molecules (so-called monovalent antigens). A trapping zone 106 with immobilized analyte analog 107, in which zone the second, analyte-free antibody 105 is trapped to avoid indication error, is therefore additionally needed in this case on the test strip 100. The participating reaction partners must be made available in exactly defined amounts. Since a specific immunological reaction takes place between the second antibody 105 and the analyte 104 in the test solution, the reaction conditions must be controlled. All this makes the process complicated, expensive, and susceptible to failure. It can hardly be performed by laymen.